Antigenic analysis of human cytomegalovirus isolates from Japanese women and infants.

The antigenic differences among human cytomegalovirus (CMV), two laboratory strains (Davis, AD 169), isolates from pregnant women's cervical secretions, mother's milk, infants' throat swabs and urine were analyzed by means of the plaque reduction assay using human sera which were assumed to contain monotypic antibodies by primary infection and boosted antibodies by reinfection or reactivation of the latent virus. In the cross-neutralization tests, there were no remarkable differences among the CMV strains examined. Moreover, in the neutralization kinetics, normalized kappa values among the strains constantly exceeded 80. Therefore, it is suggested that the human CMV strains examined in the study were serologically identical or very closely related.     

Assembly of Rickettsia tsutsugamushi progeny in irradiated L cells.

In the assembly of Rickettsia tsutsugamushi progeny in irradiated L cells, nascent forms first appear as undemarcated foci in the host cell granular cytoplasm, in which electron-lucent filamentous (f) and electron-dense granular (g) areas differentiate. Morphological observations indicated that the assembly involves formation of a filamentous network in the f area, manufacture of rickettsial ribosomes in the g area, and formation of mildly electron-dense fuzzy zones, along which a double membrane assembles.     

Growth pattern of Rickettsia tsutsugamushi in irradiated L cells.

Irradiated L cells infected with Rickettsia tsutsugamushi were studied under the electron microscope to define the morphological growth pattern of the organism. For 2 days after inoculation, no rickettsiae were found either extra- or intracellularly; after 2 days multiple rickettsiae appeared within the host cells without morphological evidence of their entry. These observations showed that the rickettsiae within the cell were assembled in situ by segregation of portions of the granular cytoplasm and subsequent internal differentiation and surface membrane assembly of the segregated bodies. The protoplasmic (P) bodies, which seemed to be formed by shedding infected-cell granular cytoplasm, consistently appeared on the surface and within the phagosomes of the host cells. Rickettsiae were occasionally seen entering host cells in the later phase of infection; these were apparently the ones assembled within the P bodies. This suggested that the P bodies, and not the rickettsiae, were the major infectious particles that transmitted the rickettsial genetic substance among the host cells. On the basis of the present morphological observations, viral-type multiplication for R. tsutsugamushi is proposed.     

Isolation of Rickettsia tsutsugamushi antigenically different from Kato, Karp, and Gilliam strains from patients

Rickettsia tsutsugamushi strains from three recent patients of Tsutsugamushi disease in Niigata Prefecture were isolated primarily in mice and then in L cell cultures. By this procedure, low virulent strains to mice, as well as high virulent ones, could be isolated and cultivated serially in L cell cultures, suggesting the usefulness of L cells for isolation of this species of rickettsia. Each newly isolated strain was identified as a member of R. tsutsugamushi from the results of cross immunological tests and morphological observation. On the other hand, it was recognized that one of these rickettsiae showed immunological properties distinguishable from the prototype strains of Kato, Karp, and Gilliam by the cross complement fixation test, and also had low virulence in mice.     

Persistence and reactivation of Rickettsia tsutsugamushi infections in laboratory mice.

The persistence of R. tsutsugamushi in tissues of experimentally infected mice for 565 days was demonstrated. Reactivation of apparently dormant infections was accomplished by inoculating the mice with a heterologous strain of the organism or treatment with cyclophosphamide.     

Antigenic and genetic variation in influenza A (H1N1) virus isolates recovered from a persistently infected immunodeficient child.

Antigenic and genetic variations have been analyzed in eight consecutive isolates recovered from a child with severe combined immunodeficiency syndrome persistently infected with naturally acquired type A (H1N1) influenza virus over a 10-month period. Hemagglutination inhibition reactions and T1 oligonucleotide fingerprinting demonstrated that these viruses were related to strains causing outbreaks in the United States at that time (1983 to 1984) but that antigenic and genetic differences between consecutive isolates could be detected. This variation between isolates was examined further by sequencing the RNAs encoding the HA1 region of the hemagglutinin (HA) and the nucleoprotein (NP) in five of the consecutive isolates. Multiple point mutations were detected in both genes, and a deletion of one amino acid was detected in the HA. Depending on the isolates compared, 5.8 x 10(-3) to 17 x 10(-3) substitutions per nucleotide site per year were detected in the RNAs encoding the HA1, and 3.5 x 10(-3) to 24 x 10(-3) substitutions per nucleotide site per year were detected in the NP gene. Fifty-four percent of the base changes in the HA1 and 73% in the NP led to amino acid substitutions. A progressive accumulation of mutations over time was not observed, suggesting that the genetic diversity of these viruses may best be interpreted as the result of shifts in the population equilibrium (quasi-species) of replicating variant genomes.     

Antigenic analysis by immunofluorescence of in vitro-produced metacyclics of Trypanosoma brucei and their infections in mice

The antigenic types in populations of Metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms with heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

Sensitivity of polymerase chain reaction assay for Rickettsia tsutsugamushi in patients' blood samples.

We developed a nested polymerase chain reaction (PCR) method to detect Rickettsia tsutsugamushi (R. tsutsugamushi) DNA and determined its sensitivity. Primers were selected from the DNA sequence of the 58-kDa group-specific antigen gene of the Karp strain. The target sequence of rickettsial DNA was detectable as the band corresponding to 88 bp in 1.0 microgram of the DNA extracted from BS-C-1 cells infected with R. tsutsugamushi. Rickettsia-specific bands were observed not only for the homologous Karp strain, but also for four heterologous strains: two other reference strains (Gilliam and Kato) and two prototype strains prevalent in Miyazaki district (Irie and Hirano). The minimum copy number detectable by this method was estimated to be five rickettsiae. All of nine peripheral blood mononuclear cell samples from patients with tsutsugamushi disease who were seen 2-11 days after disease onset tested positive for rickettsial DNA. The PCR assay method presented here could be a specific diagnostic tool for tsutsugamushi disease, especially in its early acute stage.     

Antigenic relations of Rickettsia prowazekii and Rickettsia canada, established in the study of sera of patients with Brill's disease

Sera of patients with Brill's disease and of healthy persons with spotted fever in their past history were examined in the complement fixation reaction (CFR) to determine antigenic relations between R. prowazekii and R. canada. R. canada was found to have common antigenic determinants with R. prowazekii and R. mooseri. However, the antigenic determinants of R. canada differed from those of the mentioned rickettsiae. The titres of complement-fixing antibodies in the sera of patients with Brill's disease with the antigen of R. mooseri were lower than the titres with the homologous antigen within the range of 1-2 twofold dilutions of the serum. However, the oscillations of the titres with the antigen of R. canada in the study of the same sera were expressed in 1-5 twofold dilutions. In serological identification of canada rickettsiosis, antigens of rickettsiae of the spotted fever group should invariably be included in the investigation of the sera.     

Immunological properties of Rickettsia tsutsugamushi, Kawasaki strain, isolated from a patient in Kyushu

Nine isolates of Rickettsia tsutsugamushi were obtained from patients with Tsutsugamushi disease (scrub typhus) in Miyazaki Prefecture in Kyushu. Immunological analyses of these patients' sera and the isolates were performed by indirect immunofluorescence, indirect immunoperoxidase or immunoblotting techniques. In the analysis of reactions of the patients' sera with the prototype strains Gilliam, Karp, and Kato and with the isolates, sera from two patients, including Kawasaki, showed similar profiles and cross-reaction with the two isolates recovered from the corresponding patients, but reacted only weakly with the prototype strains. With guinea pig polyclonal antibodies against the isolate and prototype strains, Kawasaki strain showed some degree of cross-reaction with Gilliam strain but not with either Karp or Kato strain, nor with Shimokoshi strain which is known to be different antigenically from the prototype strains. Additionally, strain-specific murine monoclonal antibodies against Gilliam, Karp, and Kato strains did not react at all with Kawasaki strain. These results suggest that the Kawasaki strain may be different antigenically from the prototype strains and Shimokoshi strain. The finding two strains of the same antigenic type (Kawasaki) among only nine isolates suggests the presence of Kawasaki-type rickettsiae in Miyazaki Prefecture. Shimokoshi strain also did not react with these strain-specific monoclonal antibodies, suggesting that strains of R. tsutsugamushi antigenically distinct from the prototype strains, such as Kawasaki and Shimokoshi strains, may easily be recognized by their nonreactivity with these monoclonal antibodies.     

Molecular detection of pathogens from ectoparasites recovered from peri-domestic animals,and the first description of a Candidatus Midichloria sp. from Haemaphysalis wellingtoni from rural communities in Malaysia

Rural communities in Malaysia have been shown to be exposed to Coxiella, Borrelia and rickettsial infections in previous seroprevalence studies. Further research is necessary to identify the actual causative agents and the potential vectors of these infections. The arthropods parasitizing peri-domestic animals in these communities may serve as the vector in transmitting arthropod-borne and zoonotic agents to the humans.Molecular screening of bacterial and zoonotic pathogens from ticks and fleas collected from dogs, cats and chickens from six rural communities in Malaysia was undertaken. These communities were made up of mainly the indigenous people of Malaysia, known as the Orang Asli, as well as settlers in oil palm plantations. The presence of Coxiella burnetii, Borrelia, and rickettsial agents, including Rickettsia and Anaplasma, was investigated by performing polymerase chain reaction (PCR) and DNA sequencing.Candidatus Rickettsia senegalensis was detected in one out of eight pools of Ctenocephalides felis fleas. A relapsing fever group Borrelia sp. was identified from one of seven Haemaphysalis hystricis ticks tested. The results from the PCR screening for Anaplasma unexpectedly revealed the presence of Candidatus Midichloria sp., a potential tick endosymbiont, in two out of fourteen Haemaphysalis wellingtoni ticks tested. C. burnetii was not detected in any of the samples tested.The findings here provide evidence for the presence of potentially novel strains of rickettsial and borrelial agents in which their impact on public health risks among the rural communities in Malaysia merit further investigation. The detection of a potential endosymbiont of ticks also suggest that the presence of tick endosymbionts in the region is not fully explored.     

随机扩增多态性DNA分析院内感染的铜绿假单胞菌

目的 运用随机扩增多态性DNA(RAPD)基因分型技术监测铜绿假单胞菌(PA)的医院感染情况,了解耐药表型与RAPD基因型关系,为确定院内交叉感染发生及院感控制提供依据.方法 对10例ICU患者连续多次采样分离出的48株PA进行RAPD基因分型和药物敏感性试验.结果 48株PA共分出9个基因型.6例患者为单一RAPD型PA菌株感染,4例患者检出2种RAPD 型,有3组患者分别检出同型RAPD.耐药性最高的是环丙沙星(75%),其次是左氧氟沙星(73%),耐药性最低的为美洛培南(31%).结论 耐药表型与RAPD基因型之间不存在相关性;RAPD分型快速简单,对于流行病学调查确定院内交叉感染或流行具有重要意义.     

Comparative genomics of Helicobacter pylori isolates recovered from ulcer disease patients in England

Background  

Genomic diversity of H. pylori from many different human populations is largely unknown. We compared genomes of 65 H. pylori strains from Nottingham, England. Molecular analysis was carried out to identify rearrangements within and outside the cag-pathogenicity-island (cag PAI) and DNA sequence divergence in candidate genes. Phylogenetic analysis was carried out based on various high-resolution genotyping techniques.     

Identification of two isolates of Trichinella recovered from humans in France

Two Trichinella isolates from humans in France were characterized using reproductive capacity indices and a combination of molecular methods. The isolate TRLL hybridized with the pig type-specific probe pPra and had pig type restriction profiles and rDNA patterns. It was therefore identified as a domestic or pig type isolate. The isolate CTRD-85 had similarities and differences in restriction profiles and rDNA patterns with both AF1 and Trichinella nelsoni and was identified as a sylvatic type. Pattern comparisons also show that T. nelsoni is similar to variants of the North American sylvatic type.     

The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. II. Differential Induction of Tumor Necrosis Factor-Alpha Production by Rickettsia tsutsugamushi and Rickettsia conorii

The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage-like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat-killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat-killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH-1 subset of T cells is predominant in immunity to R. tsutsugamushi.